Investigation of Antibody Tolerance in Methanol for Analytical Purposes: Methanol Effect Patterns and Molecular Mechanisms

Abstract The extraction of targets from biological samples for immunoassays using organic solvents, such as methanol, is often necessary. However, high concentrations of organic solvents in extracts invariably lead to instability of the employed antibody, resulting in poor performance of the immunoassay. Evaluating the tolerance ability and exploring the molecular mechanisms of antibody tolerance in organic solvents are essential for the development of robust immunoassays. In this work, 25 monoclonal antibodies and methanol are utilized as models to address these questions. A novel protocol is initially established to precisely and rapidly determine antibody tolerance in methanol, identifying two distinct methanol effect patterns. Through a detailed investigation of the structural basis, a novel hypothesis regarding methanol effect patterns is proposed, termed “folding‐aggregation,” which is subsequently validated through molecular dynamics simulations. Furthermore, the investigation of sequence basis reveals significant differences in residue types within the complementarity‐determining regions and ligand‐binding residues, distinguishing the two antibody methanol effect patterns. Moreover, the methanol effect patterns of the antibodies are defined by germline antibodies. This work represents the first exploration of antibody methanol effect patterns and associated molecular mechanisms, with potential implications for the discovery and engineering of tolerant antibodies for the development of robust immunoassays.